CN104211800A - Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage - Google Patents

Abstract

The present invention is directed stromal cell derived factor-1 peptides that have been mutated to make them resistant to digestion by the proteases dipeptidyl peptidase IV (DPPIV) and matrix metalloproteinase-2 (MMP-2) but which maintain the ability of native SDF-1 to attract T cells. The mutants may be attached to membranes formed by self-assembling peptides and then implanted at sites of tissue damage to help promote repair.

Description

Protease-resistant mutants of stromal cell-derived factor 1 in tissue damage repair

This application has an international application date of October 22, 2007, an international application number of PCT/US2007/022394, an application number entering the national phase of 200780039382.7, and an invention title of "Protease Antibody of Stromal Cell-derived Factor 1 in Tissue Damage Repair." A divisional application of the PCT application for "sex mutants".

Cross References to Related Applications

This application claims priority and benefit to US Provisional Application 60/929,353, filed June 22, 2007, and US Provisional Application 60/853,441, filed October 23, 2006. The contents of these prior applications are hereby incorporated by reference in their entirety.

technical field

The present invention relates to peptides of stromal cell-derived factor 1 (SDF-1) that are mutated in such a way that they retain their ability to attract cells but render them resistant to proteases, in particular matrix metalloproteinase 2 (MMP-2) and/or inactivation by dipeptidyl peptidase IV (DPPIV/CD26). These mutants promote tissue repair when they are delivered to damaged tissue. The peptides should also be effective in the treatment of a number of conditions including ulcers in the gastrointestinal tract or elsewhere, trauma from accident, surgery or disease, and cardiac tissue damage from myocardial infarction. The peptides should also be effective in the treatment of desensitizing diabetic patients to injury caused by wounds, ulcers or lesions. In a particularly preferred embodiment, the mutant form of SDF-1 is delivered to damaged tissue using a membrane formed by self-assembling peptides.

Background technique

Stromal cell-derived factor 1 (SDF-1, or CXCL12) is a 68 amino acid member of a family of chemokines that attract resting T lymphocytes, monocytes, and CD34+ stem cells. It is commonly found in two distinct forms, SDF-1α and SDF-1β, which are the result of differential mRNA splicing (US 5,563,048). These forms are essentially identical except that SDF-1β has a 4 amino acid (-Arg-Phe-Lys-Met) extension at the C-terminus. Both forms of SDF-1 begin with a 21 amino acid long signal peptide that is cleaved to form the active peptide (US 5,563,048). For the purposes of the present invention, it is understood that the term "SDF-1" refers to the active form of the peptide (ie after cleavage of the signal peptide) and encompasses SDF-1α and SDF-1β.

It was also found that the full-length 68 amino acid SDF-1 sequence is not required for activity. Peptides with at least the first 8 N-terminal residues of SDF-1 retained the receptor binding and biological activity of the full-length peptide, albeit at reduced potency. For example, SDF-1, 1-8, 1-9, 1-9 dimers, and 1-17 induce intracellular calcium and chemotaxis in T lymphocytes and CEM cells and bind to CXC chemokine receptors 4 (CXCR4). However, native SDF-1 has half-maximal chemotactic activity at 5 nM, while the 1-9 dimer requires 500 nM, thus its potency is 1/100. The potency of the 1-17 and 1-9 monomeric analogs were respectively It is 1/400 and 1/3600 of SDF-1. C-terminally cyclized variants of SDF-1 have been described that have higher binding affinity for the CXCR4 receptor, and this type of cyclization can, if desired, be used to link the peptides described in the present invention. For the purposes of the present invention, the term SDF-1 will include truncations at the C-terminus that still retain SDF-1 biological activity (i.e. chemotaxis of T lymphocytes and CEM cells and binding to CXC chemokine receptor 4 (CXCR4)) in the form of peptides. At a minimum, these truncated forms include the first 8 amino acids at the N-terminus of the peptide.

SDF-1 in hematopoiesis during embryonic development (Nagasawa et al., Nature 382: 635-638 (1996); Zou et al., Nature 393: 595-599 (1998)) and after stem cell transplantation (Lapidot et al., Blood 106: 1901-1910 (2005)) Stem cell homing to the bone marrow plays a key role. In addition to its role in stem cell homing, SDF-1 is also important in cardiogenesis as well as angiogenesis. SDF-1-deficient mice die perinatally and are defective in ventricular septal formation, bone marrow hematopoiesis, and organ-specific angiogenesis (Nagasawa et al., Nature 382:635-638 (1996); Zou et al., Nature 393:595-599( 1998)). It has also been reported that abnormally low levels of SDF-1 impair wound healing in diabetic patients, at least in part, and that this impairment can be reversed by administering this cytokine at the site of tissue injury (Gallagher et al., J. Clin Invest. 117 : 1249-1259 (2007)).

In normal adult hearts, SDF-1 is continuously expressed, but its expression is upregulated within days after myocardial infarction (Pillarisetti et al., Inflammation 25:193-300 (2001)). Askari et al. transplanted stably transfected cardiac fibroblasts overexpressing SDF-1 in the myocardium and combined with G-CSF treatment to increase the expression of SDF-1 at 8 weeks after myocardial infarction (Lancet 362: 667-703 (2003)). This was associated with higher numbers of bone marrow stem cells (c-Kit or CD34 positive) and endothelial cells in the heart and resulted in increased blood vessel density and improved left ventricular function. These studies suggest that deficiencies in naturally occurring myocardial repair processes are due in part to insufficient SDF-1 availability. Therefore, delivery of SDF-1 in a controlled manner after myocardial infarction can attract more precursor cells to promote tissue repair (Penn et al., Int. J. Cardiol. 95 (Suppl. 1): S23-S25 (2004)) . Among other things, the administration of SDF-1 can be used to improve the healing of wounds or ulcers in patients, especially diabetic patients.

One approach that can be used to sustain drug delivery at the site of tissue injury is through the application of biocompatible membranes. Certain peptides are capable of self-assembly when incubated with low concentrations of monovalent metal cations (U.S. 5,670,483; U.S. 6,548,630). Assembly results in the formation of a gel-like membrane that is nontoxic, nonimmunogenic, and relatively stable to proteases. Once formed, the membrane is stable in serum, aqueous solutions, and cell culture substrates. They can be prepared under sterile conditions, support cell growth and digest slowly when transplanted into animals. These properties make the films well suited as devices for the delivery of therapeutic agents (US20060148703 and 20060088510).

Contents of the invention

The invention is based in part on experiments based on the premise that the beneficial effects of stromal cell-derived factor 1 (SDF-1 ) in the restoration of damaged cardiac tissue are limited by high concentrations of the protease matrix metalloproteinase 2 (MMP-2) in this tissue. More specifically, we propose that MMP-2 cleaves SDF-1 and thus abolishes its ability to attract precursor cells to sites of tissue injury.

To test this premise, the inventors developed a mutant form of SDF-1 that retained the ability to attract T cells but was resistant to MMP-2 digestion. The mSDF-1 peptide adheres to specially designed membranes formed from self-assembling peptides, which are then validated in an animal model of cardiac injury. mSDF-1 adhered to the membrane and implanted in the myocardium of experimental animals was found to promote cardiac recovery to a greater extent than either SDF-1 or mSDF-1 not adhered to the membrane.

Furthermore, the inventors found that the truncated form of SDF-1 retains the same biological activity as the full-length peptide, and that mutations at the fourth or fifth amino acid protect the peptide from protease digestion.

In a first aspect of the invention, the invention relates to a mutated form of SDF-1 (mSDF-1) characterized in that the fourth and/or fifth position of the N-terminus of unmutated SDF-1 Amino acid changes (K P V S L S Y R C P C R F F E S H V A R A N V K H L K I L NT P N C A L Q I V A R L K N N N R Q V C I D P K L K W I Q E Y L E K AL N K (SEQ ID NO: 52)). Thus, the fourth amino acid is changed to a non-S amino acid and/or the fifth amino acid is changed to a non-L amino acid. As discussed above, truncated forms of the full-length SDF-1 peptide remain biologically active if the first eight amino acids (highlighted sequence in the sequence above) are present, and these truncated forms can also be modified by mutating the fourth and / or the fifth position for protease resistance. The present invention also includes these biologically active truncation mutants. Another approach is proposed, and the present invention includes peptides comprising at least amino acids 1-8 of SEQ ID NO: 52, optionally extending at the C-terminus the remaining sequence of SEQ ID NO: 52 (shown as amino acids 9-68). all or any part. In all these cases, the peptide has a sequence corresponding to that given in SEQ ID NO: 52, except that the fourth position is a non-S proteogenic amino acid and/or the fifth position is a non-L proteogenic amino acid.

For purposes of this invention, all peptide sequences are written from N-terminus (far left) to C-terminus (far right), and unless otherwise specified, all amino acids are "proteinaceous" amino acids, i.e. they are The L isomer of: alanine (A); arginine (R); asparagine (N); aspartic acid (D); cysteine (C); glutamic acid (E); Glutamine (Q); Glycine (G); Histidine (H); Isoleucine (I); Leucine (L); Lysine (K); Methionine (M); Alanine (F); Proline (P); Serine (S); Threonine (T); Tryptophan (W); Tyrosine (Y); or Valine (V). Mutant SDF-1 peptides may be abbreviated herein as "mSDF-1", "mSDF", or SDF(NqN'), where N is the one-letter name of the amino acid that was originally present, and q is its position from the N-terminus of the peptide, N' is an amino acid substituted for N. It should also be appreciated that although SEQ ID NO: 52 shows the complete full-length sequence of SDF-1α, this sequence can be extended at the C-terminus by up to four amino acids (specifically the -R-F-K-M sequence). Accordingly, the present invention includes mutant forms of SDF-1 alpha and SDF-1 beta (see US 5,563,048). In some cases, peptides mutated by adding amino acids at the N-terminus are abbreviated as "Xp-R", where X is a proteinogenic amino acid, p is an integer, and R is the peptide before extension. It should also be understood that unless otherwise specified, all pharmaceutically acceptable forms of the peptides, including all pharmaceutically acceptable salts, can be used.

The mSDF-1 peptide must retain chemotactic activity with a sensitivity (determined, for example, by the effective concentration required to obtain a 50% maximal response in the Jurkat T cell migration assay described herein) of at least 1 that of unmutated SDF-1. /10. In addition, the mSDF-1 peptide must be resistant to loss of chemotactic activity caused by matrix metalloproteinase 2 (MMP-2) cleavage. Preferably, the inactivation rate of mSDF-1 is less than 1/2 (more preferably, less than 1/4 or 1/10) the inactivation rate of SDF-1.

In one embodiment, the mSDF-1 peptide has the sequence: K P V X L S Y R C P C R FF E S H V A R A N V K H L K I L N T P N C A L Q I V A R L K N N N R QV C I D P K L K W I Q E Y L E K A L N K (SEQ ID NO: 53), wherein X is one of the 20 protein amino acids except S Any protein amino acid. The most preferred of these peptides is SDF(S4V), which has the sequence: K P V V L S Y R C P C R F F E S H V A R A N V KH L K I L N T P N C A L Q I V A R L K N N N R Q V C I D P K L K W I QE Y L E K A L N K (SEQ ID NO: 54). SEQ ID53 and 54 show the full-length sequence of the SDF-1 peptide. However, it should be understood that the truncated form of the peptide will retain activity as long as the first 8 N-terminal amino acids are present. These peptides are also part of the invention and can be rendered protease resistant by mutation of the amino acids in position 4 and/or 5.

In another embodiment, the mSDF-1 peptide has the sequence: K P V S X S Y R C P C RF F E S H V A R A N V K H L K I L N T P N C A L Q I V A R L K N N N RQ V C I D P K L K W I Q E Y L E K A L N K (SEQ ID NO: 55), wherein X is 20 proteoamino acids except L, W or any proteinogenic amino acid other than E. The most preferred of these peptides is SDF(L5P), which has the sequence: K P V S P S Y R C P C R F F E S H V A RA N V K H L K I L N T P N C A L Q I V A R L K N N N R Q V C I D P K LK W I Q E Y L E K A L N K (SEQ ID NO: 56). Furthermore, peptides that are truncated and have at least the first 8 amino acids of SEQ ID NO: 55 or 56 are included in the present invention. They may be extended at the C-terminus by additional amino acids from the sequences shown above.

The longest mSDF-1 peptide shown above has a length of 68 amino acids. However, unless otherwise specified, it is understood that additional proteinogenic amino acids may be added to the N-terminus without substantially altering chemotactic activity or MMP-2 resistance. Furthermore, the addition of the N-terminal amino acid represents a preferred embodiment because of its ability to render the peptide resistant to the second common peptidase, dipeptidyl peptidase IV (DPPIV/CD26, abbreviated herein as "DPPIV"). Digestion has the effect of resistance.

DPPIV is a 110-kD glycoprotein expressed in renal proximal tubules, enterocytes, liver, placenta, and lung, and cleaves peptides with a proline at the second position of the N-terminus (Kikawa et al., Biochim. Biophys. Acta 1751:45-51 (2005)). SDF-1 has a proline at the second position (see SEQ ID NO:52 above) and is thus cleaved by DPPIV between this proline and the following valine (Narducci et al., Blood 107:1108-1115 (2006); Christopherson, Exp. Hematol. 34:1060-1068 (2006)).

One approach to abolish the proteolytic effect of DPPIV is to alter the proline at position 2 of SDF-1 (see SEQ ID NO: 52). However, this proline is required for the biological activity of SDF-1 and thus cannot be replaced and remains a therapeutically effective peptide. However, by adding 1 to 4 amino acids (or organic groups) to the N-terminus of SEQ ID NO: 52, the activity can be maintained and a DPPIV-resistant peptide can be made. For example, it has been found experimentally that resistance to DPPIV cleavage can be obtained by adding serine to the N-terminus of the peptide.

Therefore, in another aspect, the present invention relates to the peptide _Xp -SDF-1, wherein X is preferably any proteinaceous amino acid, p is an integer from 1 to 4, and SDF-1 is represented by SEQ ID NO:52. In a preferred embodiment, n=1. It should be understood that when p is greater than 1, each of the 2-4 added amino acids can be selected from any protein amino acid described in the present invention, that is, any of these protein amino acids can appear at the first position, any species can appear in second place, and so on.

SDF-1 can also acquire DPPIV resistance by adding a "protease protective organic group" to the N-terminus. The "protease-protecting organic group" of the present invention refers to an organic group of a non-proteinogenic amino acid that, when added to the N-terminal amino acid of SDF-1, results in retention of at least 10% (and preferably at least 50% or 80%) of the chemotactic activity of unmodified SDF-1 (determined by, for example, the Jurkat T cell migration assay described in the present invention), in addition, its inactivation rate by DPPIV is less than 50% of the inactivation rate of unmodified SDF-1 % (more preferably, the rate is less than 25% or 10%). For example, X can be: R ¹ -(CH ₂ ) _d -, where d is an integer from 0 to 3, and R ¹ is selected from: hydrogen (reminder: when R ¹ is hydrogen, d must be at least 1); branched Or linear C ₁ -C ₃ alkyl; branched or linear C ₂ -C ₃ alkenyl; halogen, CF ₃ ; -CONR ⁵ R ⁴ ; -COOR ⁵ ; -COR ⁵ ; -(CH ₂ ) _q NR ⁵ R ⁴ ; -(CH ₂ ) _q SOR ⁵ ; -(CH ₂ ) _q SO ₂ R ⁵ , -(CH ₂ ) _q SO ₂ NR ⁵ R ⁴ ; and OR ⁵ , where R ⁴ and R ⁵ Each is independently hydrogen or straight or branched C ₁ -C ₃ alkyl. In case an organic group is used for X, p should be 1. Additionally, X may represent the proteinogenic amino acid discussed above, whereby 1-4 amino acids are added to SDF-1, and one or more of these added amino acids may be replaced by a protease protective organic group.

In the general formula _Xp -SDF-1, SDF-1 optionally includes any mutation at the fourth and/or fifth position of the above-mentioned SEQ ID NO:52. Accordingly, the present invention encompasses peptides of the form X _p -mSDF-1, wherein X and p are as described above, and mSDF-1 is selected from the group consisting of: SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; and SEQ ID NO: 55; ID NO: 56. These double mutant peptides are resistant to DPPIV and MMP-2.

The present invention also encompasses fusion proteins wherein any of the aforementioned mSDF-1, Xp _- SDF-1 or Xp _- mSDF-1 sequences are linked to a self-assembling peptide that forms a biocompatible film. A membrane to which protease-resistant SDF-1 is adhered may be implanted in a patient at a site of tissue injury, particularly cardiac tissue injury, trauma (whether as a result of accident, surgery, or disease) or an ulcer, and the membrane at the site Maintain the biological activity of SDF-1 for a long time. A fusion protein is formed by directly linking the C-terminus of the protease-resistant SDF-1 peptide to the N-terminus of the self-assembling peptide, or the two peptides can be linked through a linker sequence. Therefore, the present invention includes fusion proteins having the following general formula: A-(L) _n -(R) _q , wherein N is an integer from 0 to 3, q is an integer from 1 to 3, and A is the aforementioned protease-resistant SDF- 1 peptide (i.e., mSDF-1, Xp- _SDF -1 or Xp _- mSDF-1), L is a linker sequence 3-9 amino acids long, and R is a self-assembling peptide selected from :

AKAKAEAEAKAKAEAE, (SEQ ID NO: 1);

AKAEAKAEAKAEAKAE, (SEQ ID NO: 2);

EAKAEAKAEAKAEAKA, (SEQ ID NO: 3);

KAEAKAEAKAEAKAEA, (SEQ ID NO: 4);

AEAKAEAKAEAKAEAK, (SEQ ID NO: 5);

ADADARARADADARAR, (SEQ ID NO: 6);

ARADARADARADARAD, (SEQ ID NO: 7);

DARADARADARADARA, (SEQ ID NO: 8);

RADARADARADARADA, (SEQ ID NO: 9);

ADARADARADARADAR, (SEQ ID NO: 10);

ARADAKAEARADAKAE, (SEQ ID NO: 11);

AKAEARADAKAEARAD, (SEQ ID NO: 12);

ARAKADAEARAKADAE, (SEQ ID NO: 13);

AKARAEADAKARADAE, (SEQ ID NO: 14);

AQAQAQAQAQAQAQAQ, (SEQ ID NO: 15);

VQVQVQVQVQVQVQVQ, (SEQ ID NO: 16);

YQYQYQYQYQYQYQYQ, (SEQ ID NO: 17);

HQHQHQHQHQHQHQHQ, (SEQ ID NO: 18);

ANANANANANANANAN, (SEQ ID NO: 19);

VNVNVNVNVNVNVNVN, (SEQ ID NO: 20);

YNYNYNYNYNYNYNYN, (SEQ ID NO: 21);

HNHNHNHNHNHNHNHN, (SEQ ID NO: 22);

ANAQANAQANAQANAQ, (SEQ ID NO: 23);

AQANAQANAQANAQAN, (SEQ ID NO: 24);

VNVQVNVQVNVQVNVQ, (SEQ ID NO: 25);

VQVNVQVNVQVNVQVN, (SEQ ID NO: 26);

YNYQYNYQYNYQYNYQ, (SEQ ID NO: 27);

YQYNYQYNYQYNYQYN, (SEQ ID NO: 28);

HNHQHNHQHNHQHNHQ, (SEQ ID NO: 29);

HQHNHQHNHQHNHQHN, (SEQ ID NO: 30);

AKAQADAKAQADAKAQAD, (SEQ ID NO: 31);

VKVQVDVKVQVDVKVQVD, (SEQ ID NO: 32);

YKYQYDYKYQYDYKYQYD, (SEQ ID NO: 33);

HKHQHDHKHQHDHKHQHD, (SEQ ID NO: 34);

RARADADARARADADA, (SEQ ID NO: 35);

RADARGDARADARGDA, (SEQ ID NO: 36);

RAEARAEARAEARAEA, (SEQ ID NO: 37);

KADAKADAKADAKADA, (SEQ ID NO: 38);

AEAEAHAHAEAEAHAH, (SEQ ID NO: 39);

FEFEFKFKFEFEFKFK, (SEQ ID NO: 40);

LELELKLKLELELKLK, (SEQ ID NO: 41);

AEAEAKAKAEAEAKAK, (SEQ ID NO: 42);

AEAEAEAEAKAK, (SEQ ID NO: 43);

KAKAKAKAEAEAEAEA, (SEQ ID NO: 44);

AEAEAEAEAEAKAKAKAK, (SEQ ID NO: 45);

RARARARADADADADA, (SEQ ID NO: 46);

ADADADADARARARAR, (SEQ ID NO: 47);

DADADADARARARARA, (SEQ ID NO: 48);

HEHEHKHKHEHEHKHK, (SEQ ID NO: 49);

VEVEVEVEVEVEVEVEVEVE, (SEQ ID NO:50); and

RFRRFRFRFRFRFRFRFRF, (SEQ ID NO:51).

The most preferred self-assembling peptides are: RARADADARARADADA, (SEQ ID NO: 35), q=1; and the preferred protease-resistant SDF-1 peptides are SDF(S4V) and _Xp -SDF(S4V), especially p= 1. When linked together, the resulting fusion protein is, for convenience, abbreviated as SDF(S4V)-RAD or _Xp- SDF(S4V)-RAD. Preferred linker sequences arise when n = 1 and L is GGGGGG (abbreviated "6G", SEQ ID NO: 57); GIVGPL (SEQ ID NO: 58) and PVGLIG (SEQ ID NO: 59). The last represents the MMP-2 cleavage site ("MCS"). GIVGPL (SEQ ID NO: 58) represents the scrambled form of MCS, abbreviated "SCR". Surprisingly, this sequence was also found to be subject to MMP-2 cleavage, albeit at a lower rate than MCS. Preferably, the fusion protein comprising linker sequence is: SDF(S4V)-6G-RAD; Xp _- SDF(S4V)-6G-RAD; SDF(S4V)-MCS-RAD; Xp _- SDF(S4V)-MCS -RAD; SDF(S4V)-SCR-RAD; and XP-SDF(S4V)-SCR-RAD. In addition, P is preferably 1.

In another aspect, the present invention relates to nucleic acids (comprising nucleotide sequences encoding any of the aforementioned protease-resistant peptides or fusion proteins), vectors (wherein these nucleic acids are operably linked to a promoter sequence) and host cells (using the Transformation with the vector described above). The term "operably linked" means that genetic elements are linked in a manner that permits their normal function. For example, a sequence encoding a peptide is operably linked to a promoter when transcription of the peptide is under the control of the promoter and the resulting transcript is correctly translated into the peptide.

Preferred nucleic acids encoding protease resistant SDF-1 peptides and fusion proteins include:

aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaaagtggattcaggagtacctggagaaagcttaaacaag(

aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagtgaggaatcgtgggacctctgcgtgcccgtgccgacgccgacgcccgtgcccgtgccgacgccgacgcc(SEQ ID NO:61)；

aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagcctgtgggactgatcggagtgcccgtgccgacgccgacgcccgtgcccgtgccgacgccgacgcc(SEQ ID NO:62)；和

aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagggaggcgggggaggtgggcgtgcccgtgccgacgccgacgcccgtgcccgtgccgacgccgacgcc(SEQ ID NO:63)

In another aspect, the present invention relates to biocompatible membranes formed of self-assembling peptides described in published US applications 20060148703 and 20060088510 with mSDF-1, _Xp -SDF-1 or Xp _- mSDF attached -1 peptide. The term "biocompatible" means that the membrane is non-toxic and can be implanted in a patient without eliciting an immune response. Between 0.1% and 10% (and preferably 0.5-5%) of the membrane-assembled peptides bind mutated SDF-1. Binding can be covalent or non-covalent. Non-covalent binding occurs when the protease-resistant SDF-1 peptide is simply trapped into the membrane matrix and when the protease-resistant SDF-1 peptide binds to self-assembling peptides within the membrane via a biotin/avidin linkage. As used herein, the term "avidin" is intended to include streptavidin. The membrane optionally has other therapeutic agents, such as PDGF or interleukin-8, also adhered.

The use of biotin and avidin for linker molecules is well known in the art and standard methods can be applied to attach protease resistant SDF-1 peptides to self-assembling peptides, either before or after membrane formation. A specific method of linking self-assembled membranes with biotin/avidin is described in US20060088510 and this method can be applied to form membranes with adherent cytokines. To avoid steric interference between the biotin/avidin group and the protease resistant peptide, a spacer may be included between the two. The spacer can take the form of 1-15 (preferably 1-10) fatty acids or 1-15 (preferably 1-10) amino acids and separates the protease resistant SDF-1 peptide from the self-assembling peptide by at least another 12 Angstroms and up to an additional 250 Angstroms. Methods of incorporating spacers of this type are well known in the art. In a preferred embodiment, about 1% of the self-assembling peptides used in the membrane are adhered to the protease resistant SDF-1 peptide. It is also preferred that the self-assembling peptides making up the membrane are homogeneous, ie all peptides are identical.

Alternatively, the protease-resistant SDF-1 peptide can be linked to a self-assembling peptide of the membrane portion by a peptide bond, i.e. the protease-resistant SDF-1 peptide can be part of a fusion protein either directly or via an intervening linking amino acid sequence Linked to self-assembling peptides. Any of the above fusion proteins can be used, SDF(S4V)-6G-RAD; Xp _- SDF(S4V)-6G-RAD; SDF(S4V)-MCS-RAD; Xp _- SDF(S4V)-MCS-RAD; SDF(S4V)-SCR-RAD and _Xp -SDF(S4V)-SCR-RAD are particularly preferred. Said membranes are prepared from fusion proteins (or self-assembling peptides), taking advantage of the fact that the self-assembling peptides of the present invention do not aggregate together in water, but assemble in the presence of low concentrations of monovalent metal cations membrane. Thus, for example, fusion proteins can be prepared under conditions in which self-assembly does not occur, and then exposed to conditions that promote membrane formation, such as low concentrations of monovalent metal ions. Finally, a matrix that can be implanted in a patient is obtained, and it maintains a high concentration of SDF-1 biological activity at the implantation site. Alternatively, the fusion protein can be incorporated into an injectable pharmaceutical composition where the concentration of monovalent cations is too low to induce self-assembly, and then administered to a patient to induce membrane formation in vivo.

Mutated SDF-1 peptides are resistant to cleavage by MMP-2 and/or DPPIV but retain at least a portion (at least 10%, preferably more than 25%, 50% or 80%) of the chemotactic activity of native SDF-1. They are therefore ideally suited for application at sites such as damaged cardiac tissue, where MMP-2 (or DPPIV) is present in high concentrations. In addition, an MMP-2 cleavage site can (if desired) be placed within the linker region linking the SDF-1 peptide to the self-assembling peptide. This will allow the protease resistant SDF-1 peptide to be released from the grafted membrane over time.

The compositions described above should be effective in the treatment of any disease or condition characterized by high concentrations of MMP-2 and/or DPPIV, where attraction of stem cells may induce regeneration or healing. This includes the treatment of inflammatory and ischemic diseases such as stroke, limb ischemia, wound healing and diabetic ulcers. In a particularly preferred embodiment, the present invention relates to a method of treating damaged cardiac tissue, for example after a heart attack, by injecting or implanting any of the aforementioned biocompatible membranes or fusion proteins at or near the site of injury. Preferably, the membrane is injected or implanted directly into the patient's damaged tissue, such as the myocardium. The membrane should be large enough to prevent elution of protease-resistant SDF-1 by body fluids, and mSDF-1 should be present in sufficient amounts to facilitate migration of T cells to the site of injury. Guidance on these parameters is provided by the experiments described herein.

Detailed ways

The present invention is based on the concept of promoting the recovery of damaged tissue, such as damaged cardiac tissue, by exposing it to SDF-1 that has been mutated such that it is resistant to MMP-2 and/or DPPIV cleavage, and which is passed Membrane delivery of spontaneously assembled peptide formation. Such self-assembling peptides have been described in US 5,670,483 and 6,548,630 (hereby incorporated by reference in their entirety). Methods of adhering factors to membranes and the use of such membranes in delivering therapeutic agents to cardiac tissue have also been described (see Published US Applications 20060148703 and 20060088510, hereby incorporated by reference in their entirety). The same procedures for making and using membranes are applicable to the present invention.

Description of self-assembling peptides

Peptides for self-assembly should be at least 12 residues long and contain alternating hydrophobic and hydrophilic amino acids. Peptides longer than about 200 amino acids tend to have problems with solubility and membrane stability and should be avoided. Ideally, peptides should be about 12-24 amino acids in length.

Self-assembling peptides must be complementary. This means that amino acids on one peptide must have the ability to form ionic or hydrogen bonds with amino acids on another peptide. Ionic bonds can be formed between acidic amino acid side chains and basic amino acid side chains. Hydrophilic basic amino acids include Lys, Arg, His and Orn. The hydrophilic acidic amino acids are Glu and Asp. Ionic bonds can form between acidic residues of one peptide and basic residues of another peptide. The amino acids that form hydrogen bonds are Asn and Gln. Hydrophobic amino acids that can be incorporated into peptides include Ala, Val, Ile, Met, Phe, Tyr, Trp, Ser, Thr, and Gly.

Self-assembling peptides must also be "structurally compatible". This means that they must maintain a substantially constant distance from each other when combined. The inter-peptide distance for each pair of ionic or hydrogen bonds can be calculated by counting the total number of non-branched atoms on each amino acid side chain in each pair of ionic or hydrogen bonds. For example, lysine has five and glutamic acid has four unbranched atoms in the side chain. Interactions between two residues on these different peptides will result in an inter-peptide distance of 9 atoms. Within a peptide containing only EAK repeat units, all ion pairs involve lysine and glutamic acid, and thus a constant inter-peptide distance will be maintained. Therefore, these peptides will be structurally complementary. Peptides that vary by more than one atom in the distance between peptides (approximately 3-4 Angstroms) do not form gels properly. For example, if two binding peptides have an ion pair with a distance of 9 atoms and another ion pair with a distance of 7 atoms, this does not satisfy the structural complementarity requirement. A complete discussion of complementarity and structural compatibility can be found in U.S. 5,670,483 and 6,548,630.

It should also be recognized that membranes can be formed from homogeneous mixtures of peptides as well as heterogeneous mixtures of peptides. The term "homogeneous" herein means that the peptides are identical to each other. "Heterogeneous"indicates that the peptide is bound to another structurally different peptide. Regardless of whether a homogeneous or heterogeneous peptide is used, the requirements regarding amino acid alignment, length, complementarity, and structural compatibility apply. In addition, it should be recognized that the carboxyl and amino groups of the peptide terminal residues may or may not be protected using standard groups.

Peptide Preparation

The self-assembling and protease-resistant SDF-1 peptides of the invention can be prepared using standard N-tert-butoxycarbonyl (t-Boc) chemistry solid-phase peptide synthesis and using n-methylpyrrolidone chemistry cycles. Once the peptides are synthesized, they can be purified using steps such as high pressure liquid chromatography (HPLC) on reversed phase columns. Purity can also be assessed by HPLC, and the presence of the correct composition can be confirmed by amino acid analysis. Suitable purification steps for the mSDF-1 peptide are described in the Examples section.

Fusion proteins can be chemically synthesized or prepared by recombinant DNA technology. The present invention describes the full-length sequences of these proteins and provides examples of DNA sequences that can be used to generate these proteins.

SDF-1 binding to self-assembling peptides

Several strategies can be used to attach protease-resistant SDF-1 to self-assembling peptides. One strategy is non-covalent conjugation that has previously been shown to be effective for delivering PDGF-BB, a growth factor, to tissues (Hsieh et al., J. Clin. Invest. 116:237-248 (2006)).

The second adhesion strategy is the biotin-sandwich method (Davis, et al., Proc. Nat'l Acad. Sci. USA103:8155-8160 (2006)), wherein the protease-resistant SDF-1 is biotinylated and linked to the biotinylated peptide using tetravalent streptavidin as a linker. To achieve this, protease resistant SDF-1 can be coupled to a 15 amino acid sequence of an acceptor peptide (termed AP; Chen et al., Nat. Methods 2:99-104 (2005)) for biotinylation. Since the active site of SDF-1 is located near the amino-terminus, fusion proteins should be prepared by incorporating additional sequences at the C-terminus. The acceptor peptide sequence allows site-specific biotinylation by the E. coli enzyme biotin ligase (BirA; Chen et al., Nat. Methods 2:99-104 (2005)). A number of commercial kits are available for biotinylation of proteins. However, many of these reagents biotinylate lysine residues in a non-specific manner, which may reduce mSDF-1 activity, since the N-terminal lysine of SDF-1 has been shown to be critical for receptor binding as well as activity (Crump et al., EMBO J. 16:6996-7007 (1997)). Biotinylated self-assembling peptides were prepared by the MIT Biopolymer Laboratory, and, when mixed with native self-assembling peptides at a ratio of 1 to 100, the self-assembly of nanofibers should not be disturbed (Davis et al., Proc. Nat' l Acid. Sci. USA 103:8155-8160 (2006)).

A third targeted strategy is to directly incorporate protease-resistant SDF-1 peptides into self-assembling nanofibers by constructing fusion proteins of mutant SDF-1 and self-assembling peptides. For example, mSDF-1 can be coupled to the 16 amino acid sequence of SEQ ID NO:35. This "RAD" portion of the fusion protein will incorporate into the nanofibrous scaffold upon assembly.

film formation

The self-assembling peptides and fusion proteins described herein do not form films in water, but self-assemble in the presence of low concentrations of monovalent metal cations. The order of availability of these cations is Li ⁺ > Na ⁺ > K ⁺ > Cs ⁺ (US 6,548,630). Monovalent cations at a concentration of 5 mM are sufficient for peptide self-assembly, and concentrations as high as 5M are still effective. The anion associated with the monovalent metal cation is not critical to the invention and may be acetate, chloride, sulfate, phosphate, and the like.

The starting concentration of the self-assembling peptide will affect the final size and thickness of the formed film. In general, the higher the peptide concentration, the higher the degree of film formation. Formation can occur at peptide concentrations as low as 0.5 mM or 1 mg/ml. However, membranes are preferably formed at higher peptide starting concentrations, such as 10 mg/ml, to facilitate better handling properties. In general, adding the peptide to the salt solution produces better membrane formation than adding the salt to the peptide solution.

Film formation is relatively independent of pH and temperature. However, the pH should be kept below 12 and the temperature should generally be in the range of 4-90°C. Divalent metal cations at or above 100 mM lead to incorrect film formation and should therefore be avoided. Similarly, sodium lauryl sulfate at concentrations of 0.1% or higher should be avoided.

Film formation can be observed by simple visual inspection, assisted if necessary with a dye such as Congo Red. Membrane integrity can also be observed microscopically, with or without staining.

Pharmaceutical Composition and Dosage

Membranes of Adnectinase-resistant SDF-1 peptides or fusion proteins can be incorporated into pharmaceutical compositions containing carriers such as saline, water, Ringer's solution, and other agents or excipients. Dosage forms are usually designed for implantation or injection, especially into cardiac tissue, but topical treatments can also be effective, for example in trauma treatment. All dosage forms can be prepared by methods standard in the art (see, eg, Remington's Pharmaceutical Sciences , 16th ed. A. Oslo. ed., Eason, PA (1980)).

Those skilled in the art are expected to adjust the dose on a case-by-case basis using well-established methods of clinical medicine. Optimal dosages are determined by methods known in the art and are influenced by factors such as patient age, disease state, and other clinically relevant factors.

Example

Example 1: Biological Effects and Protease Resistance of SDF-1 Mutants

SDF-1 purification and expression

The DNA sequence of mature SDF-la can be cloned from human cDNA into the pET-Sumo vector, and an additional N-terminal serine residue can be incorporated to facilitate cleavage by Sumo protease (generating the 69 amino acid form of SDF-1). Fusion proteins can be prepared by incorporating RAD or AP sequences in reverse transcription primers. The Sumo-SDF-1 fusion protein can be expressed in Rosetta DE3 E. coli and grown at 37°C to an optical density (600nm) of 1.5. Cells were induced with 0.25 mM isopropyl β-D-thiogalactoside for 4 hours and harvested by centrifugation. As described below, SDF-1α can be purified by a 3-step process, all steps performed at 21°C.

Cells from the 4-L growth were lysed and homogenized in 300 ml lysis buffer (6M guanidine, 20 mM phosphate (pH 7.8), 500 mM NaCl). Debris was collected by centrifugation at 3000g. The first purification step consists of capturing the polyhistidine tag present in the Sumo-SDF-1α fusion protein with Nickel-NTA. The nickel-NTA resin was washed with washing buffer (8M urea, 500mM NaCl, 20mM phosphate (pH6.2)), and the bound protein was eluted at pH4. Further purification and oxidative refolding was carried out on a cation exchange HPLC column. Samples were adjusted to binding buffer (8M urea, 30mM 2-mercaptoethanol, 1mM EDTA, 50mM Tris pH 8) and loaded onto an HPLC column. Refolding of Sumo-SDF-1 was performed on the column by running for 2 hours in refolding buffer (50mM Tris pH 8, 75mM NaCl, 0.1mM reduced glutathione and 0.1mM oxidized glutathione). Sumo-SDF-1 was eluted and concentrated by step gradient (0.5 to 1M NaCl). The SUMO-SDF-1 fusion protein was cleaved by Sumo protease 1 (1U/50μg protein) in 50mM Tris pH8.0, 500mM NaCl. Samples were adjusted to 0.1% trifluoroacetic acid (TFA) and loaded on a C18 reverse phase HPLC column for the final purification step. The column was subjected to a linear gradient from 30% to 40% acetonitrile in 0.1% TFA. Fractions containing SDF-1 were lyophilized and resuspended. The activity of purified SDF-1 was tested by migration of the Jurkat T lymphocyte line.

Modifications of SDF-1 constructs

The SDF-1 fusion construct was modified by insertional mutagenesis of one of three sequences: one sequence sensitive to MMP-2 cleavage (MMP cleavage site or MCS), and the other sequence containing the same amino acids but in a random order ( scrambled sequence or SCR), the third sequence contains 6 glycines as a linker.

MMP cleavage site mutations in chemokines

SDF-1 is cleaved at its N-terminal active site by MMP-2, yielding the N-terminal tetrapeptide and inactivated SDF-1(5-68). To confer resistance to cleavage by MMP-2 to SDF-1, specific mutations of four different amino acids were made based on the MMP-2 substrate sequence described by Netzel-Arnett et al. (Biochemtstry 32:6427-6432 (1993)) . These four different constructs were expressed and purified as described above for SDF-1. Among the four different mutations, SDF-1(L5W) and SDF-1(L5E) showed the least activity on T cell migration. In contrast, SDF-1(S4V) and SDF-1(L5P) showed comparable biological activity to native SDF-1. Because SDF-1(L5P) is more difficult to purify, SDF-1(S4V) was chosen for further experiments.

Effects of Mutations on Protease Sensitivity and Chemotactic Activity

Mutant forms of SDF-1 were detected at a concentration of 100 nM in the Jurkat T cell migration assay. This experiment showed that both SDF-1(S4V) and SDF-1(L5P) retained most of the activity of unmutated SDF-1 in promoting T cell migration. This activity was greatly reduced in the SDF-1(L5W) mutant and the SDF-1(L5E) mutant.

The sensitivity of the peptides to MMP-2 cleavage was determined by incubating the mutants with the enzyme for 1 hour and then examining the incubation products by SDS-PAGE. This assay revealed that, unlike SDF-1, the mutant did not undergo a shift in position indicative of cleavage. MMP-2 incubation was found to reduce the chemotactic activity of SDF-1, but not SDF-1(S4V), as shown in the Jurkat T cell migration assay. These results suggest that the S4V variant of SDF-1 retains chemokine bioactivity but resists activation by MMP-2.

In vivo data

A blinded randomized study was performed to evaluate the effect of different SDF-1 forms on cardiac function after myocardial infarction in rats. Ejection fraction was measured with a Millar catheter system for measuring intraventricular pressure as well as ventricular volume. Both SDF-1(S4V)-6G-RAD and SDF-1(S4V)-MCS-RAD significantly improved cardiac function four weeks after myocardial infarction in rats compared to the MI-only group. This suggests that MMP-2 resistance (SDF-1(S4V)) and adhesion to membranes are required for successful cardiac repair therapy.

Example 2: Experiments with truncated forms of SDF-1

Three truncated forms of SDF-1 are commercially synthesized; all include the first 17 amino acids of native SDF-1. Two mutants of the SDF-117 amino acid were designed to be more resistant to MMP-2 based on our previous work on the intact SDF-1 protein:

SDF-117AA:

KPVSLSYRCPCRFFESH (SEQ ID 64)

SDF-1(S4V)17AA:

KPVVLSYRCPCRFFESH (SEQ ID 65)

SDF-1(L5P)17AA:

KPVSPSYRCPCRFFESH (SEQ ID 66)

Migration experiments were performed with the Jurkat T lymphocyte line. The truncated SDF-1 17AA is 1/500 as potent as native SDF-1, but induces a similar maximal migration as native SDF-1. Therefore, the same T lymphocyte migration response should be observed if a concentration 500-fold compared to the full-length protein is used. The potency of mutated SDF-1(S4V)17AA and SDF-1(L5P)17AA was 1/3 that of SDF-117AA without mutation. This is an alteration similar to that seen between native SDF-1 and SDF-1(S4V).

Cleavage experiments were performed on the peptides using MMP-2: 2 nmoles of SDF-117AA, SDF-1(S4V)17AA and SDF-1(L5P)17AA were incubated with MMP-2 for 1 hour at RT. Proteins run on SDS-PAGE showed cleavage of SDF-1 17AA but not SDF-1(S4V)17AA or SDF-1(L5P)17AA. Therefore, these truncated proteins are therapeutically effective because they are still biologically active and also MMP-2 resistant.

Documents cited herein are fully incorporated by reference. Now that the invention has been fully described, those skilled in the art should understand that the invention can be practiced within a broad and equivalent range of conditions, parameters, etc., without affecting the spirit or scope of the invention or any embodiment thereof.

Sequence Listing

the

<110> The Brigham and Womens' Hospital, Inc.

Lee, Richard

Segers, Vincent

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Arg Ala Arg Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp Ala

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Arg Ala Glu Ala Arg Ala Glu Ala Arg Ala Glu Ala Arg Ala Glu Ala

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Ala Glu Ala Glu Ala Glu Ala Glu Ala Lys Ala Lys

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<400> 48

the

Asp Ala Asp Ala Asp Ala Asp Ala Arg Ala Arg Ala Arg Ala Arg Ala

1 5 10 15

the

the

<210> 49

<211> 16

<212> PRT

<213> Artificial

the

<220>

<223> Synthetic sequences designed for self-assembly

the

<400> 49

the

His Glu His Glu His Lys His Lys His Glu His Glu His Lys His Lys

1 5 10 15

the

the

<210> 50

<211> 20

<212> PRT

<213> Artificial

the

<220>

<223> Synthetic sequences designed for self-assembly

the

<400> 50

the

Val Glu Val Glu Val Glu Val Glu Val Glu Val Glu Val Glu Val Glu

1 5 10 15

the

the

Val Glu Val Glu

                       

the

the

<210> 51

<211> 20

<212> PRT

<213> Artificial

the

<220>

<223> Synthetic sequences designed for self-assembly

the

<400> 51

the

Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe

1 5 10 15

the

the

Arg Phe Arg Phe

                       

the

the

<210> 52

<211> 68

<212> PRT

<213> Homo sapiens

the

<400> 52

the

Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

20 25 30

the

the

Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

35 40 45 45

the

the

Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60 60

the

the

Ala Leu Asn Lys

65

the

the

<210> 53

<211> 68

<212> PRT

<213> Homo sapiens

the

the

<220>

<221> misc_feature

<222> (4)..(4)

<223> X is ala, arg, asx, cys, glx, gly, his, ile, leu, lys, met, phe,

pro, thr, trp, tyr, or val

the

<400> 53

the

Lys Pro Val Xaa Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

20 25 30

the

the

Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

35 40 45 45

the

the

Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60 60

the

the

Ala Leu Asn Lys

65

the

the

<210> 54

<211> 68

<212> PRT

<213> Homo sapiens

the

<400> 54

the

Lys Pro Val Val Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

20 25 30

the

the

Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

35 40 45 45

the

the

Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60 60

the

the

Ala Leu Asn Lys

65

the

the

<210> 55

<211> 68

<212> PRT

<213> Homo sapiens

the

the

<220>

<221> misc_feature

<222> (5)..(5)

<223> X is ala, arg, asx, cys, gln, gly, his, ile, lys, met, phe, pro,

ser, thr, tyr, or val

the

<400> 55

the

Lys Pro Val Ser Xaa Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

20 25 30

the

the

Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

35 40 45 45

the

the

Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60 60

the

the

Ala Leu Asn Lys

65

the

the

<210> 56

<211> 68

<212> PRT

<213> Homo sapiens

the

<400> 56

the

Lys Pro Val Ser Pro Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

20 25 30

the

the

Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

35 40 45 45

the

the

Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60 60

the

the

Ala Leu Asn Lys

65

the

the

<210> 57

<211> 6

<212> PRT

<213> Homo sapiens

the

<400> 57

the

Gly Gly Gly Gly Gly Gly Gly

1 5 5

the

the

<210> 58

<211> 6

<212> PRT

<213> Homo sapiens

the

<400> 58

the

Gly Ile Val Gly Pro Leu

1 5 5

the

the

<210> 59

<211> 6

<212> PRT

<213> Homo sapiens

the

<400> 59

the

Pro Val Gly Leu Ile Gly

1 5 5

the

the

<210> 60

<211> 204

<212> DNA

<213> Homo sapiens

the

<400> 60

aagcccgtcg tcctgagcta cagatgccca tgccgattct tcgaaagcca tgttgccaga 60

the

gccaacgtca agcatctcaa aattctcaac actccaaact gtgcccttca gattgtagcc 120

the

cggctgaaga acaacaacag acaagtgtgc attgacccga agctaaagtg gattcaggag 180

the

tacctggaga aagctttaaa caag 204

the

the

<210> 61

<211> 273

<212> DNA

<213> Homo sapiens

the

<400> 61

aagcccgtcg tcctgagcta cagatgccca tgccgattct tcgaaagcca tgttgccaga 60

the

gccaacgtca agcatctcaa aattctcaac actccaaact gtgcccttca gattgtagcc 120

the

cggctgaaga acaacaacag acaagtgtgc attgacccga agctaaagtg gattcaggag 180

the

tacctggaga aagctttaaa caagtgagga atcgtgggac ctctgcgtgc ccgtgccgac 240

the

gccgacgccc gtgcccgtgc cgacgccgac gcc 273

the

the

<210> 62

<211> 269

<212> DNA

<213> Homo sapiens

the

<400> 62

aagcccgtcg tcctgagcta cagatgccca tgccgattct tcgaaagcca tgttgccaga 60

the

gccaacgtca agcatctcaa aattctcaac actccaaact gtgcccttca gattgtagcc 120

the

cggctgaaga acaacaacag acaagtgtgc attgacccga agctaaagtg gattcaggag 180

the

tacctggaga aagctttaaa caagcctgtg ggactgatcg gagtgcccgt gccgacgccg 240

the

acgcccgtgc ccgtgccgac gccgacgcc 269

the

the

<210> 63

<211> 270

<212> DNA

<213> Homo sapiens

the

<400> 63

aagcccgtcg tcctgagcta cagatgccca tgccgattct tcgaaagcca tgttgccaga 60

the

gccaacgtca agcatctcaa aattctcaac actccaaact gtgcccttca gattgtagcc 120

the

cggctgaaga acaacaacag acaagtgtgc attgacccga agctaaagtg gattcaggag 180

the

tacctggaga aagctttaaa caagggaggc gggggaggtg ggcgtgcccg tgccgacgcc 240

the

gacgcccgtg cccgtgccga cgccgacgcc 270

the

the

<210> 64

<211> 17

<212> PRT

<213> Homo sapiens

the

<400> 64

the

Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His

the

the

the

<210> 65

<211> 17

<212> PRT

<213> Homo sapiens

the

<400> 65

the

Lys Pro Val Val Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His

the

the

the

<210> 66

<211> 17

<212> PRT

<213> Homo sapiens

the

<400> 66

the

Lys Pro Val Ser Pro Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

the

the

His

Claims (13)

1. An isolated mutant stromal cell-derived factor 1 (SDF-1) peptide, comprising general formula mSDF-1 or _Xp -mSDF-1, wherein said SDF-1 is at least amino acid 1 comprising SEQ ID NO:52 A peptide of the amino acid sequence of -8, and it optionally extends all or any part of the remaining sequence of SEQ ID NO:52 at the C-terminus, and the remaining sequence of said SEQ ID NO:52 is shown as amino acid 9-68, and wherein : a) X is a protein amino acid or a protease protective organic group; b) p is an integer between 1 and 4; c) mSDF-1 is a mutant form of said SDF-1 comprising a mutation of the fourth and/or fifth amino acid at the N-terminal of said SDF-1, wherein mSDF-1 has chemotactic activity for T cells, and its Inactivation by matrix metalloproteinase 2 (MMP-2) at less than half the rate of native SDF-1 peptide; and d) _Xp -mSDF-1 is a mutated form of said SDF-1 comprising a mutation of the fourth and/or fifth amino acid at the N-terminal of said SDF-1, wherein _Xp -mSDF-1 has T cell Chemotactic activity, which is inactivated by dipeptidyl peptidase IV (DPPIV) at a rate less than half that of native SDF-1 and which is inactivated by MMP-2 at a rate less than native SDF-1 half of the inactivation rate. 2. The isolated mutant SDF-1 peptide of claim 1, comprising said mSDF-1 peptide or said _Xp -mSDF-1 peptide, wherein said mSDF-1 peptide or said _Xp -mSDF-1 peptide: a) comprising the amino acid sequence of SEQ ID NO:53; b) maintain chemotactic activity for T cells which is at least 10% of native SDF-1; and c) The rate of inactivation by MMP-2 is less than one-fourth that of native SDF-1. 3. The isolated mutant SDF-1 peptide of claim 1 comprising the general formula of said mSDF-1 peptide or said _Xp -mSDF-1 peptide, wherein said mSDF-1 peptide or said _Xp -mSDF- 1 peptide: a) comprising the amino acid sequence of SEQ ID NO:55; b) maintain chemotactic activity for T cells which is at least 10% of native SDF-1; and c) The rate of inactivation by MMP-2 is less than one-fourth that of native SDF-1. 4. A fusion protein comprising the general formula A-(L) _n -(R) _q , wherein: A is the isolated mutant SDF-1 peptide of claim 1, n is an integer of 0-3, and q is an integer of 1-3 Integer, L is a linker sequence of 3-9 amino acids, and R is a self-assembling peptide selected from the group consisting of: AKAKAEAEAKAKAEAE (SEQ ID NO: 1); AKAEAKAEAKAEAKAE (SEQ ID NO: 2); EAKAEAKAEAKAEAKA (SEQ ID NO: 3); KAEAKAEAKAEAKAEA (SEQ ID NO: 4); AEAKAEAKAEAKAEAK (SEQ ID NO: 5); ADADARARADADARAR (SEQ ID NO: 6); ARADARADARADARAD (SEQ ID NO: 7); DARADARADARADARA (SEQ ID NO: 8); ADARADARADARADAR (SEQ ID NO: 10); ARADAKAEARADAKAE (SEQ ID NO: 11); AKAEARAKAEARAD (SEQ ID NO: 12); ARAKADAEARAKADAE (SEQ ID NO: 13); AKARAEADAKARADAE (SEQ ID NO: 14); AQAQAQAQAQAQAQAQ (SEQ ID NO: 15); VQVQVQVQVQVQVQVQ (SEQ ID NO: 16); YQYQYQYQYQYQYQYQ (SEQ ID NO: 17); HQHQHQHQHQHQHQHQ (SEQ ID NO: 18); ANANANANANANANAN (SEQ ID NO: 19); VNVNVNVNVNVNVNVN (SEQ ID NO: 20); YNYNYNYNYNYNYNYN (SEQ ID NO: 21); HNHNHNHNHNHNHNHN (SEQ ID NO: 22); ANAQANAQANAQANAQ (SEQ ID NO: 23); AQANAQANAQANAQAN (SEQ ID NO: 24); VNVQVNVQVNVQVNVQ (SEQ ID NO: 25); VQVNVQVNVQVNVQVN (SEQ ID NO: 26); YNYQYNYQYNYQYNYQ (SEQ ID NO: 27); YQYNYQYNYQYNYQYN (SEQ ID NO: 28); HNHQHNHQHNHQHNHQ (SEQ ID NO: 29); HQHNHQHNHQHNHQHN (SEQ ID NO: 30); AKAQADAKAQADAKAQAD (SEQ ID NO: 31); VKVQVDVKVQVDVKVQVD (SEQ ID NO: 32); YKYQYDYKYQYDYKYQYD (SEQ ID NO: 33); HKHQHDHKHQHDHKHQHD (SEQ ID NO: 34); RADARGDARADARGDA (SEQ ID NO: 36); RAEARAEARAEARAEA (SEQ ID NO: 37); KADAKADAKADAKADA (SEQ ID NO: 38); AEAEAHAHAEAEAHAH (SEQ ID NO: 39); FEFEFKFKFEFEFKFK (SEQ ID NO: 40); LELELKLKLELELKLK (SEQ ID NO: 41); AEAEAKAKAEAEAKAK (SEQ ID NO: 42); AEAEAEAEAKAK (SEQ ID NO: 43); KAKAKAKAEAEAEAEA (SEQ ID NO: 44); AEAEAEAEAEAKAKAKAK (SEQ ID NO: 45); RARARARADADADADA (SEQ ID NO: 46); ADADADADARARARAR (SEQ ID NO: 47); DADADADARARARA (SEQ ID NO: 48); HEHEHKHKHEHEHKHK (SEQ ID NO: 49); VEVEVEVEVEVEVEVEVEVE (SEQ ID NO: 50); and RFRFRFRFRFRFRFRFRFF (SEQ ID NO: 51). 5. The fusion protein of claim 4, wherein A is said mSDF-1 peptide or said Xp _- mSDF-1 peptide, and comprises the amino acid sequence of any one of SEQ ID NO:53-SEQ ID NO:56, particularly wherein A is the mSDF-1 peptide of SEQ ID NO:54. 6. The fusion protein of claim 5, wherein n=1, and L is selected from the group consisting of: GGGGGG (SEQ ID NO:57); GIVGPL (SEQ ID NO:58) and PVGLIG (SEQ ID NO:59). 7. A nucleic acid comprising a nucleotide sequence encoding the isolated mutant stromal cell-derived factor 1 of claim 1 or the fusion protein of claim 4. 8. A biocompatible peptide film comprising one or more peptides selected from the group consisting of SEQ ID NO:1-SEQ ID NO:8, SEQ ID NO:10-SEQ ID NO:34, or SEQ ID NO:36 - A self-assembling peptide of the amino acid sequence of SEQ ID NO: 51, and wherein 0.1-10% of said self-assembling peptide is bound to the isolated mutant stromal cell-derived factor 1 of claim 1. 9. The biocompatible peptide film of claim 8, wherein the isolated mutant stromal cell-derived factor 1 is bound to self-assembling peptides within the film via a biotin/avidin linkage, or covalently via a peptide bond. Bind to self-assembling peptides within the membrane. 10. The biocompatible peptide film of claim 9, wherein a spacer separates said isolated mutant stromal cell-derived factor 1 from said one or more self-assembling peptides by at least 14 Angstroms and no more than 250 Angstroms . 11. Use of the mutated stromal cell-derived factor 1 of claim 1 in the preparation of a medicament for treating a patient to assist repair of damaged tissue, said treatment comprising: locally applying the mutated stromal cell-derived factor 1 of claim 1 to the said damaged tissue, particularly wherein said patient is treated for a disease or condition selected from the group consisting of: stroke; limb ischemia; tissue damage due to trauma; and diabetic ulcers. 12. The use of claim 11, wherein said mutant stromal cell-derived factor 1 is adhered to a biocompatible membrane or to a self-assembling peptide that forms a biological Compatible film. 13. The use of claim 11, wherein said patient is being treated for damage to cardiac tissue, and said treatment comprises injecting or implanting said biocompatible peptide membrane into said patient's myocardium.